Growth factor complex

ABSTRACT

An isolated protein complex is provided which includes a growth factor, growth factor binding protein and vitronectin. Preferably, the isolated protein complex includes an insulin-like growth factor-I, insulin-like growth factor binding protein-3 or insulin-like growth factor binding protein-5 and vitronectin. Also provided are methods of modulating cell proliferation and/or migration by administering said protein complex for the purposes of wound healing, skin repair and tissue replacement therapy. Conversely, by using agents that disrupt growth factor protein complexes formed in vivo, growth factor-driven cell proliferation and/or migration may be suppressed such as for the purposes of treating cancers, psoriasis, atherosclerosis and wounds prone to hypertrophic scarring.

FIELD OF THE INVENTION

THIS INVENTION relates to an isolated protein complex which includes agrowth factor binding protein and vitronectin. In particular, thisinvention relates to an isolated protein complex which includes aninsulin-like growth factor, an insulin-like growth factor bindingprotein and vitronectin. Also provided by the invention are growthfactor complexes comprising variant growth factors and/or growth factorbinding proteins that facilitate or enhance formation of the growthfactor complexes. This invention also provides methods of modulatingcell proliferation and/or migration by administering said proteincomplex for the purposes of wound healing, skin repair, cosmetic skinmaintenance and tissue replacement therapy. Conversely, by disruptingprotein complexes formed ill vivo, growth factor-driven cellproliferation and/or migration can be suppressed such as for thepurposes of treating cancers, psoriasis, atherosclerosis and woundsprone to hypertrophic scarring. These treatments may have medical andveterinary applications.

BACKGROUND OF THE INVENTION

Skin growth, repair and healing are subject to complex biologicalcontrol mechanisms which act via both positive and negative signals.Such signals act at the level of controlling cell proliferation,differentiation and migration, and are typically mediated by growthfactor polypeptides. In this regard, important growth factors includeepidermal growth factor (EGF) and insulin-like growth factors (IGF-I and-II).

Human IGF-I has been reported to exert a wide range of biologicalactivities including stimulation of cell proliferation, differentiationand migration, protection from protein degradation and apoptosis, aswell as regulation of endocrine factors such as growth hormone. IGF-IIhas similar properties to IGF-I but appears to be more relevant tocarcinogenesis and fetal and embryonic development, IGF-I having agreater role in postnatal development.

Both IGF-I and IGF-II act through a binding interaction with the type IIGF receptor (IGFR). The availability of the IGFs for such aninteraction is regulated by insulin like growth factor binding proteins(IGFBPs 1-6). IGFBPs are known to both positively and negativelyregulate IGF function as well as exhibit IGF-independent activity.

Another functional component of IGF pathways is the type II IGFR whichis also known as the cation-independent mannose-6-phosphate receptor(CI-MPR). The type II IGFR is a multifunctional protein that bindslysosomal enzymes bearing mannose-6-phosphate moieties, as well asIGF-II, although the functional significance of IGF-]1 binding isunclear (O'Dell & Day, 1998, Int. J. Biochem. Cell Biol. 30 767;Braulke, 1999, Horn. Metab. Res. 31 10 242; Nykjaer et al., 1998, J.Cell. Biol. 141 815).

The IGFs have also been reported to bind another group of proteinstermed “IGFBP-related proteins” which share structural similarity andinclude connective tissue growth factor (CTGF) and products encoded bythe mac25, nov and cyr61 genes. These bind IGFs with much lower affinitythan do IGFBPs. More recently, vitronectin (VN) has been identified asan extracellular matrix protein, structurally unrelated to IGFBPs andIGFBP-related proteins, that binds IGF-II but not IGF-I (Upton et al.,1999, Endocrinol. 140 2928).

Vitronectin is an ˜75 kD, glycosylated extracellular matrix proteinwhich is also found in blood, and has been implicated in cancers, bonediseases and pathological disorders involving angiogenesis (reviewed inSchvartz et al., 1999, Int. J. Biochem. Cell Biol. 31 539). The role ofvitronectin in events such as angiogenesis and tumorigenesis at leastpartly resides in the ability of vitronectin to bind integrins and tointeract with components of the urokinase plasminogen activator system(for example PAI-1, uPAR, plasminogen) to thereby promote cellproliferation, adhesion, spreading and migration. Vitronectin has morespecifically been implicated in preventing tumor cell apoptosis inresponse to drug treatment (Uhm et al., 1999, Clin. Cancer Res. 5 1587).Vitronectin appears to be a carrier of IGF-II in the circulation(McMurtry et al., 1996, J. Endocrinol. 150 149).

OBJECT OF THE INVENTION

The present inventor has surprisingly discovered that IGFBPs bindvitronectin, and that IGF-I can bind vitronectin when bound to an IGFBP.

It is therefore an object of the invention to provide an isolated IGFBPand vitronectin-containing complex.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides an isolated polypeptidecomplex comprising a growth factor binding protein and vitronectin.

Preferably, the isolated polypeptide complex further comprises a growthfactor.

A preferred growth factor is insulin-like growth factor-I (IGF-I).

Other growth factors include epidermal growth factor (EGF), fibroblastgrowth factor (FGF), basic fibroblast growth factor (bFGF), osteopontin,thrombospondin-1, tenascin-C, PAI-1, plasminogen, fibrinogen, fibrin andtransferrin.

A preferred growth factor binding protein is an insulin-like growthfactor binding protein selected from the group consisting of IGFBP1 ,IGFBP2, IGFBP3, IGFBP4, IGFBP5 and IGFBP6.

A more preferred insulin-like growth factor binding protein is IGFBP2,IGFBP3, IGFBP4 or IGFBP5.

An even more preferred insulin-like growth factor binding protein isIGFBP3 or IGFBP5.

In a second aspect, the invention provides an isolated protein complexcomprising an IGFBP-related protein and vitronectin.

Preferably, the isolated polypeptide complex further comprises a growthfactor, preferably IGF-I.

In one embodiment, the IGFBP-related protein is selected from the groupconsisting of connective tissue growth factor (CTGF), a polypeptideencoded by the mac25 gene, a polypeptide encoded by the nov gene and apolypeptide encoded by the cyr61 gene.

In a third aspect, the invention provides a isolated protein complexcomprising vitronectin, a variant growth factor and/or a variant growthfactor binding protein.

In one embodiment, the isolated protein complex of this aspect comprisesvitronectin and a variant growth factor engineered to include a heparinbinding domain (HBD).

In another embodiment, the isolated protein complex of this aspectcomprises vitronectin and a non-glycosylated growth factor bindingprotein.

In yet another embodiment, the isolated protein complex of this aspectcomprises vitronectin and a variant growth factor selected from thegroup consisting of des(1-6)IGF-II and des(1-3)IGF-I.

Also contemplated according to the first- second- and third-mentionedaspects is that the isolated protein complex of the invention mayfurther include an acid-labile subunit, a polypeptide which can complexwith an IGFBP, referred to hereinafter as ALS.

The invention according to the first-, second- and third-mentionedaspects also contemplates isolated protein complexes comprising variantsand biologically-active fragments of growth factors, growth factorbinding proteins, IGFBP-related proteins and vitronectin, and use ofsuch complexes. Biologically-active fragments and variants includewithin their scope analogues, mutants, agonists and antagonists of saidgrowth factors, growth factor binding proteins, IGFBP-related proteinsand vitronectin.

In a fourth aspect, the invention provides a pharmaceutical compositioncomprising one or more isolated protein complexes according to thefirst-, second- or third-mentioned aspect and apharmaceutically-acceptable carrier or diluent.

In a fifth aspect, the invention provides a pharmaceutical compositioncomprising a expression construct comprising one or more nucleic acidsencoding an isolated protein complex according to the first-, second- orthird-mentioned aspect and a pharmaceutically-acceptable carrier ordiluent.

In a sixth aspect, the invention provides a transformed cell capable ofexpressing a recombinant protein complex, or recombinant proteinscapable of forming said complex, according to the first-, second- orthird-mentioned aspects.

In a seventh aspect, the invention provides a method of modulating cellproliferation and/or migration including the step of administering to ananimal or isolated cells thereof, an isolated protein complex accordingto the first-, second- or third-mentioned aspects.

Preferably, the isolated protein complex comprises an IGFBP andvitronectin.

Preferably, the isolated protein complex further comprises IGF-I.

In an eighth aspect, the invention provides a method of modulating cellproliferation and/or migration, including the step of administering toan animal or isolated cells thereof an agent which prevents or disruptsformation of a protein complex according to the first-, second- orthird-mentioned aspects.

Preferably, the agent prevents or disrupts an interaction between anIGFBP and vitronectin.

More preferably, the agent prevents or disrupts an interaction betweenIGFBP and vitronectin wherein the protein complex comprises IGF-I.

The agent may be an antagonist of an interaction between an IGFBP andvitronectin or between an IGFBP-related protein and vitronectin, forexample.

An example of an agent that inhibits formation of IGFBP and vitronectincomplexes is IGF-II.

As will be described in more detail hereinafter, disruption ofinteractions between vitronectin and IGFBPs may not only inhibit tumorcell proliferation, but also inhibit tumor metastasis, both events beingcentral to tumor pathology.

Further aspects of the invention provide uses of the isolated proteincomplexes and methods according to the aforementioned aspects of theinvention in therapeutic or prophylactic treatments of diseasesinvolving epithelial cells such as psoriasis, atherosclerosis,deterioration of the gastrointestinal epithelium and epithelial breastcancer, and/or in treatments which promote wound healing, skin repair,ulcer and burn healing, in vitro skin regeneration such as for graftingof autologous skin, bone regeneration and repair of damaged neuronaltissue.

Accordingly, the invention also provides a surgical implant orprosthesis comprising an isolated protein complex of the invention. Thesurgical implant or prosthesis may be coated, impregnated or otherwisepretreated with said isolated protein complex.

The animal treated according to the invention may be a mammal,preferably a human, or may be a non-mammalian vertebrate such as a fish,reptile or bird, or cells isolated from any of these.

Throughout this specification, unless otherwise indicated, “comprise”,“comprises” and “comprising” are used inclusively rather thanexclusively, so that a stated integer or group of integers may includeone or more other non-stated integers or groups of integers.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

TABLE 1: List of references disclosing nucleic acids encoding growthfactors and growth factor binding proteins.

TABLE 2: IGF-I and IGFBP-5 bound to vitronectin stimulates proteinsynthesis in HaCAT human keratinocytes. Data are derived frommeasurements of ³H-leucine incorporation and are expressed as %stimulation above control (without IGF-I, IGFBP-5 or vitronectin) over24 hr from a single experiment in which each treatment was tested intriplicate. IGF-I was added to IGFBP-5 (5 ng/well) in the presence (+)of vitronectin (300 ng/well) or in the absence (−) of vitronectin.

FIG. 1: Competition binding assay using increasing concentrations ofeither insulin (▴) or IGF-II (♦)to compete with [¹²⁵I]-IGF-II forbinding to 300 ng vitronectin (VN) per well. Radiolabeled IGF-II (10,000cpm) was added to VN-coated wells and the number of counts bound wasdetermined after overnight incubation and several washes. The binding isexpressed as a percentage of the binding observed in control wells withno IGF-II or insulin added. The results are shown as the average ofthree replicates±standard deviation from a representative of threeexperiments.

FIG. 2: Competition binding assay using increasing concentrations ofeither IGF-I or IGF-II to compete with [¹²⁵I]-IGF-II for binding to 300ng vitronectin (VN) per well. Radiolabeled IGF-II (10,000 cpm) was addedto either VN (▪)—or IGFBP2 (♦)-coated wells and the number of countsbound was determined after overnight incubation and several washes. Thebinding is expressed as a percentage of the binding observed in controlwells with no IGF added.

FIG. 3: Competition binding assay using increasing concentrations ofeither proIGF-II (▪) or IGF-II (♦) to compete with [¹²⁵I]-IGF-II forbinding to 300 ng vitronectin (VN) per well. Radiolabeled IGF-II (10,000cpm) was added to vitronectin-coated wells and the number of countsbound determined after an overnight incubation and several washes. Thebinding is expressed as a percentage of the binding observed in thecontrol wells with no added IGF-II or proIGF-II. The results are shownas the average of three replicates±SEM from two separate experiments.

FIG. 4: Competition binding assay using increasing concentrations ofeither PAI-I (▪) or IGF-II (♦) to compete with [¹²⁵I]-IGF-II for bindingto 300 ng of vitronectin (VN) per well. Radiolabeled IGF-II (10,000 cpm)was added to vitronectin-coated well and the number of counts bound wasdetermined after an overnight incubation and several washes. The bindingis expressed as a percentage of the binding observed in the controlwells with no added IGF-II or PAI-I. The results are shown as theaverage of three replicates±standard deviation from a representative ofthree separate experiments.

FIG. 5: Competition binding assay comparing effect of preincubation of(A) IGF-II and IGFBP3 and (B) IGF-I and IGFBP3 upon binding tovitronectin. IGFBP3 was non-glycosylated and produced in E. coli.Increasing concentrations of IGFBP3 plus either 10,000 cpm [¹²⁵I]-IGF-II(A) or [¹²⁵I]-IGF-I (B) were preincubated for 4 hr and added tovitronectin-coated wells. Alternatively, IGFBP3 plus either 10,000 cpm[¹²⁵I]-IGF-II (A) or [¹²⁵I]-IGF-I (B) were added to vitronectin-coatedwells without preincubation. The binding is expressed as the cpmobtained in the absence of non-specific binding. The results are shownas the average of three replicates from a representative of threeseparate experiments.

FIG. 6: Binding of labelled IGF-I to VN-coated wells in the presence of:(A) IGFBP1, (B) IGFBP2, (C) IGFBP3, (D) IGFBP4, (E) IGFBP5 and (F)IGFBP6. The recombinant IGFBPs were produced in mammalian cells. Thedata, expressed as average cpm of labelled IGF-I bound/VN-coated well(300 ng/well) in the presence of the indicated IGFBP are from sixindividual determinations. Ten thousand cpm of radiolabelled IGF-I wasadded to each well.

FIG. 7: Binding of labelled IGF-I to VN in the presence of “Gly IGFBP-3”(glycoslyated IGFBP-3), “IGFBP-3 HBD mutant” (IGFBP-3 with the putativeheparin binding domain mutated) and to “non-gly IGFBP-3”(non-glycosylated IGFBP-3).

FIG. 8: Competition binding assay using increasing concentrations ofIGFs and deslGFs to compete with non-glycosylated IGFBP3 incubated with[¹²⁵I]-IGF-I or -II. 30 ng (A) or 10 ng (B) of IGFBP3 plus 10,000 cpm[¹²⁵I]-IGF-II (A) or IGF-I (B) were added to VN-coated wells. Increasingconcentrations of IGF-I, IGF-II, des (1-3) IGF-I (not shown) anddes(1-6) IGF-II were added to compete with the radiolabel for binding toVN. Control wells coated with VN were treated with either 10 ng (A) or30ng (B) IGFBP3 are shown together with control wells coated with VNalone. The binding is expressed as cpm obtained in the absence ofnon-specific binding. The results are expressed as the average of threereplicates±standard deviation from a representative of three separateexperiments.

FIG. 9: Stimulation of protein synthesis in human keratinocytes. Thedata, expressed as % stimulation above control (-VN, -IGF-II) over 24 his pooled from three replicate experiments in which each treatment wastested in triplicate. The theoretical additive effect represented byopen bars (effect of VN alone) combined with grey bars (effect of IGF-IIalone) is compared with the actual observed effect of IGF-II prebound toVN (black bars). In all instances except the lowest concentration ofIGF-II tested, the actual observed effect is significantly greater(p<0.05) than the calculated additive effect.

FIG. 10: Binding of labelled IGF-II to VN-coated wells in the presenceof:

(A) IGFBP1, (B) IGFBP2, (C) IGFBP3, (D) IGFBP4, (E) IGFBP5 and (F)IGFBP6. The recombinant IGFBPs were produced in mammalian cells. Thedata, expressed as average cpm of labelled IGF-II bound/VN-coated well(300 ng/well) in the presence of the indicated IGFBP are from sixindividual determinations. Ten thousand cpm of radiolabelled IGF-II wasadded to each well.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has arisen, at least in part, from the discoveryby the present inventors that IGF-I binds vitronectin via a bindinginteraction between an IGFBP and vitronectin. Furthermore, the presentinvention describes variant IGFs and IGFBPs that may be used to augmentor diminish binding between IGFS, IGFBPs and vitronectin. Thesediscoveries have led the present inventors to manipulate these bindinginteractions in vitro with a view to manipulating contingent in vivobiological events associated with cell growth, proliferation andmigration. This invention therefore has utility in medical treatmentssuch as wound healing, skin repair and maintenance, bone regeneration,atherosclerosis and cancer therapy in both medical and veterinary areas.

For the purposes of this invention, by “isolated ” is meant removed froma natural state or otherwise subjected to human manipulation. Isolatedmaterial may be substantially or essentially free from components thatnormally accompany it in its natural state, or may be manipulated so asto be in an artificial state together with components that normallyaccompany it in its natural state.

By “polpeptide” is also meant “protein”, either term referring to anamino acid polymer.

Proteins and peptides inclusive of growth factor, growth factor bindingproteins and vitronectin proteins may be isolated in native, chemicalsynthetic or recombinant synthetic form.

A “peptide” is a protein having no more than fifty (50) amino acids.

A “biologically-active fragment” is a fragment, portion or segment of aprotein which displays at least 1%, preferably at least 10%, morepreferably at least 25% and even more preferably at least 50% of thebiological activity of the protein.

Peptides may be readily synthesized by recombinant or chemical synthesistechniques. For example, reference may be made to solution synthesis orsolid phase synthesis as described, for example, in Chapter 9 entitled“Peptide Synthesis” by Atherton and Shephard which is included in apublication entitled “Synthetic Vaccines” edited by Nicholson andpublished by Blackwell Scientific Publications. Peptide synthesismethods are also described in Chapter 18 of CURRENT PROTOCOLS IN PROTEINSCIENCE Eds. Coligan et al. (John Wiley & Sons NY, 1997) which isincorporated herein by reference. Alternatively, peptides can beproduced by digestion of a polypeptide of the invention with proteinasessuch as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease.The digested fragments can be purified by, for example, high performanceliquid chromatographic (HPLC) techniques.

In a preferred form, the invention provides an isolated protein complexcomprising vitronectin, a growth factor and a growth factor bindingprotein.

By “growth factor” is meant a molecule that stimulates or promotesgrowth of an organism or cells of said organism. A preferred growthfactor is a protein or peptide that stimulates cell division, and inparticular, mammalian cell division.

Preferably, isolated protein complexes of the invention comprise thegrowth factor IGF-I.

Isolated IGF and IGFBP polypeptides are commercially available fromsources such as GroPep (Adelaide, Australia), while VN polypeptides arecommercially available from sources such as Promega Corporation (MadisonWis. USA). Recombinant IGFs, IGFBPs and VN are readily made by personsskilled in the art, as will be discussed in more detail hereinafter.

As will be appreciated by the skilled person, the invention alsoincludes precursor forms of IGFs. Examples of pro-IGP-I proteins areIGF-I proteins that have had the signal peptide removed but are notfully processed by cleavage of the E-domain. IGF-I has three different Edomains that result from differential mRNA splicing and these precursorproteins may be present in isolated protein complexes of the invention.

However, the invention also contemplates isolated protein complexescomprising a growth factor such as epidermal growth factor (FGF;

(Heldin et al., 1981, Science 4 1122-1123), fibroblast growth factor(FGF;

Nurcombe et al., 2000, J. Biol. Chem. 275 30009-30018), basic fibroblastgrowth factor (bFGF; Taraboletti et al., 1997, Cell Growth. Differ. 8471-479), osteopontin (Nam et al., 2000, Endocrinol. 141 1100),thrombospondin-I (Nam et al., 2000, supra), tenascin-C (Arai et al.,1996, J. Biol. Chem. 271 6099), PAI-I (Nam et al., 1997, Endocrinol. 1382972), plasminogen (Campbell et al., 1998, Am. J. Physiol. 275 E321),fibrinogen (Campbell et al., 1999, J. Biol. Chem 274 30215), fibrin(Campbell et al., 1999, supra) or transferrin (Weinzimer et al., 2001,J. Clin. Endocrinol. Metab. 86 1806).

Preferably, isolated protein complexes comprising osteopontinthrombospondin-1, tenascin-C or PAI-1 further comprise IGPBP-5.

Preferably, isolated protein complexes comprising plasminogenfibrinogen, fibrin or transferin further comprise IGFBP-3.

The invention contemplates isolated protein complexes comprisingmonomeric and multimeric vitronectin, as vitronectin can exist inmonomeric and mutimeric states. In particular, multimeric VN accumulatesin areas of vascular injury and also is the predominant form of VN intissue. Thus the multimeric form of VN provides the opportunity to forma VN complex in which more than one type of growth factor or growthfactor binding protein can be delivered at the same time.

It will also be appreciated by the skilled person that isolated proteincomplexs of the invention may include vitronectin in “native”,“denatured” or “extended” states as are well understood in the art.

The invention also contemplates isolated growth factor complexescomprising nectinepsin, which is an extracellular matrix protein thatshows 60% homology to VN at the amino acid level (Blanchert et al.,1996, J. Biol. Chem. 271 26220-26226).

It will also be understood that variants of growth factors, growthfactor binding proteins and/or vitronectin may be used to form isolatedprotein complexes of the invention and may be useful in the methods ofuse set forth herein.

As used herein,“variant” proteins, polypeptides and peptides of theinvention include those in which one or more amino acids have beenreplaced by different amino acids.

It is well understood in the art that some amino acids may be changed toothers with broadly similar properties without changing the nature ofthe activity of the polypeptide (conservative substitutions).

Substantial changes in function may be made by selecting substitutionsthat are less conservative. Other replacements would be non-conservativesubstitutions and relatively fewer of these may be tolerated. Generally,the substitutions which are likely to produce the greatest changes in apolypeptide's properties are those in which (a) a hydrophilic residue(e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue(e.g., Ala, Leu, Ile, Phe or Val); (b) a cysteine or proline issubstituted for, or by, any other residue; (c) a residue having anelectropositive side chain (e.g., Arg, His or Lys) is substituted for,or by, an electronegative residue (e.g., Glu or Asp) or (d) a residuehaving a bulky side chain (e.g., Phe or Trp) is substituted for, or by,one having a smaller side chain (e.g., Ala, Ser)or no side chain (e.g.,Gly).

Variants also include proteins, polypeptides and peptides which havebeen altered, for example by conjugation or complexing with otherchemical moieties or by post-translational modification techniques aswould be understood in the art. Such derivatives include amino aciddeletions and/or additions.

Other polypeptide and peptide variants contemplated by the inventioninclude, but are not limited to, modification to side chains,incorporation of unnatural amino acids and/or their derivatives duringpeptide, polypeptide or protein synthesis and the use of crosslinkersand other chemicals which impose conformational constraints on thepolypeptides and peptide variants of the invention, Examples of sidechain modifications contemplated by the present invention includemodifications of amino groups such as by acylation with aceticanhydride; acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; amidination with methylacetimidate;carbamoylation of amino groups with cyanate; pyridoxylation of lysinewith pyridoxa1-5-phosphate followed by reduction with NaBH₄; reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; and trinitrobenzylation of amino groups with 2, 4,6-trinitrobenzene sulphonic acid (TNBS).

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitization, by way ofexample, to a corresponding amide.

The guanidine group of arginine residues may be modified by formation ofheterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

Sulphydryl groups may be modified by methods such as performic acidoxidation to cysteic acid; formation of mercurial derivatives using4-chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate;2-chloromercuri-4-nitrophenol, phenylmercury chloride, and othermercurials; formation of a mixed disulphides with other thiol compounds;reaction with maleimide, maleic anhydride or other substitutedmaleimide; carboxymethylation with iodoacetic acid or iodoacetamide; andcarbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified, for example, by alkylation of theindole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides orby oxidation with N-bromosuccinimide.

Tyrosine residues may be modified by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

The imidazole ring of a histidine residue may be modified byN-carbethoxylation with diethylpyrocarbonate or by alkylation withiodoacetic acid derivatives.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include but are not limited to, use of 4-amino butyricacid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine,norvaline, phenylglycine, omithine, sarcosine, 2-thienyl alanine and/orD-Isomers of amino acids.

Modifications also include within their scope O- and N-linkedglycosylation variants and non-glycosylated forms of proteins that, intheir naturally-occurring state, are glycosylated.

With regard to variants, these may be created by mutagenizing apolypeptide or by mutagenizing an encoding nucleic acid, such as byrandom mutagenesis or site-directed mutagenesis. Examples of nucleicacid mutagenesis methods are provided in in Chapter 9 of CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al., supra which isincorporated herein by reference.

It will be appreciated by the skilled person that site-directedmutagenesis is best performed where knowledge of the amino acid residuesthat contribute to biological activity is available. In many cases, thisinformation is not available, or can only be inferred by molecularmodeling approximations, for example.

In such cases, random mutagenesis is contemplated. Random mutagenesismethods include chemical modification of proteins by hydroxylamine (Ruanet al., 1997, Gene 188 35), incorporation of dNTP analogs into nucleicacids (Zaccolo et al., 1996, J. Mol. Biol. 255 589) and PCR-based randommutagenesis such as described in Stemmer, 1994, Proc. Natl. Acad. Sci.USA 91 10747 or Shafikhani et al., 1997, Biotechniques 23 304, each ofwhich references is incorporated herein. It is also noted that PCR-basedrandom mutagenesis kits are commercially available, such as theDiversify™ kit (Clontech).

The invention also contemplates use of growth factor variants such asdes(1-6)IGF-II and des(1-3)IGF-I to form isolated protein complexes ofthe invention.

Other variant IGFs and IGFBPs useful as agonists or antagonists will bedescribed in more detail hereinafter.

Recombinant Growth Factor Complexes

It will be appreciated that isolated protein complexes may be producedusing recombinant growth factors, growth factor binding proteins and/orvitronectin, by expression of an encoding nucleic acid in an appropriatehost cell or in a cell-free expression system as are well known in theart.

The term “nucleic acid” as used herein designates single-ordouble-stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA andgenomic DNA.

A “polynucleotide” is a nucleic acid having eighty (80) or morecontiguous nucleotides, while an “oligonucleotide” has less than eighty(80) contiguous nucleotides.

A “probe” may be a single or double-stranded oligonucleotide orpolynucleotide, suitably labeled for the purpose of detectingcomplementary sequences in Northern or Southern blotting, for example.

A “primer” is usually a single-stranded oligonucleotide, preferablyhaving 15-50 contiguous nucleotides, which is capable of annealing to acomplementary nucleic acid “template” and being extended in atemplate-dependent fashion by the action of a DNA polymerase such as Taqpolymerase, RNA-dependent DNA polymerase or Sequenase™.

Nucleic acids encoding IGFs, EGF, IGFBPs, ALS and VN are well known inthe art and have been available for many years. However, the skilledperson is referred to Table 1 which lists references that provideexamples of these nucleic acid sequences. All references in Table 1 areincorporated herein by reference.

Nucleic acids useful according to the present invention may be preparedaccording to the following procedure:

-   -   (i) creating primers which are, optionally, degenerate wherein        each comprises a respective portion of a target nucleic acid;        and    -   (ii) using said primers in combination with a nucleic acid        amplification technique to amplify one or more amplification        products from a nucleic acid extract.

Suitable nucleic acid amplification techniques are well known to theskilled person, and include polymerase chain reaction (PCR) as forexample described in Chapter of Ausubel et al. supra, which isincorporated herein by reference; strand displacement amplification(SDA) as for example described in U.S. Pat. No 5,422,252 which isincorporated herein by reference; rolling circle replication (RCR) asfor example described in Liu et al., 1996, J. Am. Chem. Soc. 118 1587,International application WO92/01813 and International ApplicationWO97/19193 which are incorporated herein by reference; nucleic acidsequence-based amplification (NASBA) as for example described bySooknanan et al.,1994, Biotechniques 17 1077 which is incorporatedherein by reference; ligase chain reaction (LCR) as for exampledescribed in International Application W089/09385 which is incorporatedby reference herein; and Q-β replicase amplification as for exampledescribed by Tyagi et al., 1996, Proc. Natl. Acad. Sci. USA 93 5395which is incorporated herein by reference.

As used herein, an “anplification product” refers to a nucleic acidproduct generated by nucleic acid amplification techniques.

Recombinant proteins may be prepared by any suitable procedure known tothose of skill in the art.

For example, the recombinant protein may be prepared by a procedureincluding the steps of:

-   -   (i) preparing an expression construct which comprises a nucleic        acid, operably linked to one or more regulatory nucleotide        sequences;    -   (ii) transfecting or transforming a suitable host cell with the        expression construct; and    -   (iii) expressing the polypeptide in said host cell.

For the purposes of host cell expression, the recombinant nucleic acidis operably linked to one or more regulatory sequences in an expressionvector.

An “expression vector” may be either a self-replicatingextra-chromosomal vector such as a plasmid, or a vector that integratesinto a host genome.

By “operably linked” is meant that said regulatory nucleotidesequence(s) is/are positioned relative to the recombinant nucleic acidof the invention to initiate, regulate or otherwise controltranscription.

Regulatory nucleotide sequences will generally be appropriate for thehost cell used for expression. Numerous types of appropriate expressionvectors and suitable regulatory sequences are known in the art for avariety of host cells.

Typically, said one or more regulatory nucleotide sequences may include,but are not limited to, promoter sequences, leader or signal sequences,ribosomal binding sites, transcriptional start and terminationsequences, translational start and termination sequences, and enhanceror activator sequences.

Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter.

In a preferred embodiment, the expression vector contains a selectablemarker gene to allow the selection of transformed host cells. Selectablemarker genes are well known in the art and will vary with the host cellused.

The expression vector may also include a fusion partner (typicallyprovided by the expression vector) so that the recombinant polypeptideof the invention is expressed as a fusion polypeptide with said fusionpartner. The main advantagse of fusion partners are that they assistidentification and/or purification of said fusion polypeptide and alsoenhance protein expression levels and overall yeild.

In order to express said fusion polypeptide, it is necessary to ligate anucleotide sequence according to the invention into the expressionvector so that the translational reading frames of the fusion partnerand the nucleotide sequence of the invention coincide.

Well known examples of fusion partners include, but are not limited to,glutathione-S-transferase (GST), Fc potion of human IgG, maltose bindingprotein (MBP) and hexahistidine (HIS₆), which are particularly usefulfor isolation of the fusion polypeptide by affinity chromatography. Forthe purposes of fusion polypeptide purification by affinitychromatography, relevant matrices for affinity chromatography areglutathione-, amylose-, and nickel- or cobalt-conjugated resinsrespectively. Many such matrices are available in “kit” form, such asthe QLAexpress™ system (Qiagen) useful with (HIS₆) fusion partners andthe Pharmacia GST purification system.

Another fusion partner well known in the art is green fluorescentprotein (GFP). This fusion partner serves as a fluorescent “tag” whichallows the fusion polypeptide of the invention to be identified byfluorescence microscopy or by flow cytometry. The GFP tag is useful whenassessing subcellular localization of the fusion polypeptide of theinvention, or for isolating cells which express the fusion polypeptideof the invention. Flow cytometric methods such as fluorescence activatedcell sorting (FACS) are particularly useful in this latter application.

In some cases, the fusion partners also have a protease cleavage site,such as for Factor X_(a) or Thrombin, which allow the relevant proteaseto partially digest the fusion polypeptide of the invention and therebyliberate the recombinant polypeptide of the invention therefrom. Theliberated polypeptide can then be isolated from the fusion partner bysubsequent chromatographic separation.

Fusion partners according to the invention also include within theirscope “epitope tags”, which are usually short peptide sequences forwhich a specific antibody is available. Well known examples of epitopetags for which specific monoclonal antibodies are readily availableinclude c-myc, influenza virus haemagglutinin and FLAG tags.

The recombinant protein may be conveniently prepared by a person skilledin the art using standard protocols as for example described inSambrook, et al., MOLECULAR CLONING. A Laboratory Manual (Cold SpringHarbor Press, 1989), incorporated herein by reference, in particularChapter 16 and 17;

CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley& Sons, Inc. 1995-1999), incorporated herein by reference, in particularChapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds.Coligan et al., (John Wiley & Sons, Inc. 1995-1999) which isincorporated by reference herein, in particular Chapters 1, 5 and 6.

In one embodiment, recombinant expression of growth factor, growthfactor binding protein and vitronectin may be performed separately, andcomplexes formed therefrom.

In another embodiment, recombinant expression of growth factor, growthfactor binding protein and vitronectin may be performed in the samecell, and complexes formed therefrom.

As hereinbefore, polypeptides of the invention may be produced byculturing a host cell transformed with said expression constructcomprising a nucleic acid encoding a polypeptide, or polypeptidehomolog, of the invention. The conditions appropriate for proteinexpression will vary with the choice of expression vector and the hostcell. This is easily ascertained by one skilled in the art throughroutine experimentation.

Suitable host cells for recombinant expression include bacteria such asE. coli, Clostridium sp., Pseudomonas sp., yeast, plant cells, insectcells (such as Sf9) and mammalian cells such as fibroblasts andkeratinocytes.

Preferred host cells are human keratinocytes.

Inducible and non-Inducible expression vectors are contemplated.

In stably transfected mammalian cells, a number of inducible andrepressible systems have been devised including metallothionine (MT)inducible and tetracycline and repressible (tetR), each of which iscontemplated by the present invention.

Particular examples of suitable expression vectors and methods ofrecombinant IGFBP expression may be found in U.S. Pat. No. 5,973,115which is incorporated herein by reference.

Minietics, Agonists and Antagonists

The invention contemplates agents which may promote, prevent or disruptformation of protein complexes comprising growth factors, growth factorbinding proteins and vitronectin. Such an agent may be a mimetic. Theterm “mimetic” is used herein to refer to molecules that are designed toresemble particular functional regions of proteins or peptides, andincludes within its scope the terms “agonist”, “analogue” and“antagonise” as are well understood in the art.

Of relevance is the elucidation of the portions of IGFs and IGFBPs whichare responsible for IGF-IGFBP binding, as described in InternationalPublication WO00/23469. Furthermore, agonist variants of IGF-I have beenmade which selectively bind IGFBP-1 or IGFBP-3, as described inInternational Publication WO00/40612.

It is therefore contemplated that agents could be engineered whichdisrupt or prevent formation of polypeptide complexes between IGFBPs andVN.

An example would be a peptide which competes for binding of the IGFBP toVN by resembling the binding site on VN or the IGFBP.

As will be described in more detail hereinafter, IGF-II is an agent thatcan inhibit binding between an IGFBP and vitronectin. It is alsoproposed that residues V³⁵ or S³⁶ along with S³⁹ of the RVSRRSR sequenceat positions 34-40 in the C-domain of IGF-II could be mutated to basicresidues to thereby resemble the HBD of IGFBP3 (BXBBB wherein B is abasic amino acid residue). This would create an agent even more capableof inhibiting formation of a complex between IGFBPs and vitronectin.

An example of an agonist contemplated by the present invention is IGF-1engineered to include a heparin binding domain (HBD) of an IGFBP tothereby bind vitronectin directly. For example, the sequence SSSRRAPQTin the C-domain of IGF-I may be engineered to have a BXBBB motif. Apreferred BXBBB motif is the KGRKR sequence of IGFBP-3 (residues228-232).

Alternatively, the putative vitronectin-binding domain of IGF-II:

RVSRRSR (residues 34-40) may be introduced into IGF-I.

An example of an antagonist of the invention is an IGFBP engineered tomutate basic residues in the HBD (as hereinbefore described) to reduceor prevent binding of the IGFBP to vitronectin.

Suitably, the engineered IGFBP is capable of binding IGF-1.

Preferably, the engineered IGFBP is IGFBP-3 or IGFBP-5.

It is also contemplated that an analogue of an IGFBP could be engineeredwhich enables formation of a complex between the analogue and VN.

Suitably, the analogue would also bind an IGF. Potential advantages ofsuch an analogue is that it might be more readily synthesized orisolated than an IGFBP, have a particular desired biological half-lifeand perhaps be engineered to specifically bind IGF-I.

The aforementioned mimetic may be peptides, polypeptides or otherorganic molecules, preferably small organic molecules, with a desiredbiological activity and half-life.

Computer-assisted structural database searching is becoming increasinglyutilized as a procedure for identifying mimetics. Database searchingmethods which, in principle, may be suitable for identifying mimetics,may be found in International Publication WO 94/18232 (directed toproducing HIV antigen mimetics), U.S. Pat. No. 5,752,019 andInternational Publication WO 97/41526 (directed to identifying EPOmimetics), each of which is incorporated herein by reference.

Other methods include a variety of biophysical techniques which identifymolecular interactions. These allow for the screening of candidatemolecules according to whether said candidate molecule affects formationof IGF-IGFBP-VN complexes, for example. Methods applicable topotentially useful techniques such as competetive radioligand bindingassays (see Upton et al., 1999, supra for a relevant method), analyticalultracentrifugation, microcalorimetry, surface plasmon resonance andoptical biosensor-based methods are provided in Chapter 20 of CURRENTPROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons,1997) which is incorporated herein by reference.

Pharmaceutical Compositions

The invention includes administration of a protein complex of theinvention in the form of a pharmaceutical composition. Pharmaceuticalcompositions of the invention may include isolated protein complexesthat comprise variant IGFs and/or IGFBPs or agents that disrupt orprevent formation of said complexes as hereinbefore described.

Suitably, the pharmaceutical composition comprises apharmaceutically-acceptable carrier. Pharmaceutical compositions mayalso include polypeptide variants, fragments or mimetics as hereinbeforedefined.

By “pharmaceutically-acceptable carrier” is meant a solid or liquidfiller, diluent or encapsulating substance that may be safely used insystemic administration. Depending upon the particular route ofadministration, a variety of carriers, well known in the art may beused. These carriers may be selected from a group including sugars,starches, cellulose and its derivatives, malt, gelatine, talc, calciumsulfate, vegetable oils, synthetic oils, polyols, alginic acid,phosphate buffered solutions, emulsifiers, isotonic saline, andpyrogen-free water.

Any suitable route of administration may be employed for providing apatient with the composition of the invention. For example, oral,rectal, parenteral, sublingual, buccal, intravenous, intra-articular,intramuscular, intra-dermal, subcutaneous, inhalational, intraocular,intraperitoneal, intracerebroventricular, transdermal and the like maybe employed. Intra-muscular and subcutaneous injection is appropriate,for example, for administration of immunogenic compositions, vaccinesand DNA vaccines.

Dosage forms include tablets, dispersions, suspensions, injections,solutions, syrups, troches, capsules, suppositories, aerosols,transdermal patches and the like. These dosage forms may also includeinjecting or implanting controlled releasing devices designedspecifically for this purpose or other forms of implants modified to actadditionally in this fashion. Controlled release of the therapeuticagent may be effected by coating the same, for example, with hydrophobicpolymers including acrylic resins, waxes, higher aliphatic alcohols,polylactic and polyglycolic acids and certain cellulose derivatives suchas hydroxypropylmethyl cellulose. In addition, the controlled releasemay be effected by using other polymer matrices, liposomes and/ormicrospheres.

Pharmaceutical compositions of the present invention suitable for oralor parenteral administration may be presented as discrete units such ascapsules, sachets or tablets each containing a pre-determined amount ofone or more therapeutic agents of the invention, as a powder or granulesor as a solution or a suspension in an aqueous liquid, a non-aqueousliquid, an oil-In-water emulsion or a water-In-oil liquid emulsion.

With regard to pharmaceutical compositions comprising IGF and IGFBPs,particular reference is made to U.S. Pat. No. 5,936,064 andInternational Publications WO99/62536 and WO99/54359 which areincorporated herein by reference.

Pharmaceutical compositions of the invention may also include expressionvectors such as viral vectors such as vaccinia, and viral vectors usefulin gene therapy. The latter include adenovirus and adenovirus-associatedviruses (AAV) such as described in Braun-Falco et al.,1999, Gene Ther. 6432, retroviral and lentiviral vectors such as described in Buchshacheret al., 2000, Blood 95 2499 and vectors derived from herpes simplexvirus and cytomegalovirus. A general overview of viral vectors useful inendocrine gene therapy is provided in Stone et al., 2000, J. Endocrinol.164 103.

The present invention may also utilize specific expression vectors whichtarget gene expression to epidermal cells, such as described in U.S.Pat. No. 5,958,764 and for in vivo wound healing applications, such asdescribed in U.S. Pat. No. 5,962,427.

Each of the aforementioned publications is incorporated herein byreference.

Therapeutic Uses

The invention provides methods of treatment using polypeptide complexesof the invention. These methods are particularly aimed at therapeutictreatment of mammals, and more particularly, humans.

Such methods include administration of pharmaceutical compositions ashereinbefore defined, and may be by way of microneedle injection intospecific tissue sites, such as described in U.S. Pat. No. 6,090,790,topical creams, lotions or sealant dressings applied to wounds, burns orulcers, such as described in U.S. Pat. No. 6,054,122 or implants whichrelease the composition such as described in International PublicationWO99/47070.

Gene therapy is also applicable in this regard, such as according tomethods set forth in U.S. Pat. No. 5,929,040 and U.S. Pat. No.5,962,427.

There also exist methods by which skin cells can be genetically modifiedfor the purpose of creating skin substitutes, such as by geneticallyengineering desired growth factor expression (Supp et al., 2000, J.Invest. Dennatol. 114 5). An example of a review of this field isprovided in Bevan et al., Biotechnol. Gent. Eng. Rev. 16 231.

Also contemplated is “seeding” a recipient with transfected ortransformed cells, such as described in International PublicationWO99/11789 .

These methods can be used to stimulate cell proliferation and therebyfacilitate or progress wound and burn healing, repair of skin lesionssuch as ulcers, tissue replacement and grafting such as by in vitroculturing of autologous skin, re-epithelialization of internal organssuch as kidney and lung and repair of damaged nerve tissue.

Skin replacement therapy has become well known in the art, and mayemploy use of co-cultured epithelial/keratinocyte cell lines, forexample as described in Kehe et al., 1999, Arch. Dermatol. Res. 291 600or in vitro culture of primary (usually autologous) epidermal, dermaland/or keratinocyte cells. These techniques may also utilize engineeredbiomaterials and synthetic polymer “scaffolds”.

Examples of reviews of the field in general are provided in Terskikh &Vasiliev, 1999, Int. Rev. Cytol. 188 41 and Eaglestein & Falanga, 1998,Cutis 62 1.

More particularly, the production of replacement oral mucosa useful incraniofacial surgery is described in Izumi et al., 2000, J. Dent. Res.79 798. Fetal keratinocytes and dermal fibroblasts can be expanded invitro to produce skin for grafting to treat skin lesions, such asdescribed in Fauza et al., J. Pediatr. Surg. 33 357, while skinsubstitutes from dermal and epidermal skin elements cultured in vitro onhyaluronic acid-derived biomaterials have been shown to be potentiallyuseful in the treatment of burns (Zacchi et al., 1998, J. Biomed. Mater.Res. 40 187).

Another aspect of epithelial cell therapy relates to healing of theepithelial lining of the gastrointestinal tract to treat or preventimpaired gut function.

Polymer scaffolds are also contemplated for the purpose of facilitatingreplacement skin engineering, as for example described in Sheridan etal., 2000, J. Control Release 14 91 and Fauza et al., 1998, supra, asare microspheres as agents for the delivery of slin cells to wounds andburns (LaFrance & Armstrong, 1999, Tissue Eng. 5 153).

The aforementioned techniques may be readily utilized according to thepresent invention by use of isolated protein complexes of the inventionto promote skin cell proliferation for the purposes of tissuereplacement and for cosmetic skin treatments.

With regard to bone regeneration, the invention provides surgical orprosthetic implants coated, impregnated or otherwise pretreated with anisolated protein complex of the invention.

Conversely, inhibition or suppression of cell proliferation andmigration by preventing or disrupting formation of IGF-IGFBP-VNcomplexes may constitute a prophylactic or therapeutic treatment ofpsoriasis or malignancies such as epithelial cancers such as breastcancer.

The invention also contemplates a method of differentiating a stem orprogenitor cell by administering an isolated protein complex of theinvention to said stem or progenitor cell. Isolated protein complexescomprising variant growth factors and IGFBPs may also be applicable tothis method.

Differentiated cells produced according to this method may be useful intherapeutic methods such as as hereinbefore described.

For example, smooth muscle cells are considered to be “mesenchymal stemcells” and may be driven to differentiate into fibroblasts, stromalcells, endothelial cells, bone or adipocytes.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting examples.

All competitive radioligand binding assays as described herein wereperformed essentially as described in Upton et al., 1999, supra.

EXAMPLE 1

Competition Binding Assay to Assess Ability of Insulin, pro-IGF-If andIGF-I to Compete with IGF-II for Binding to Vitronectin

Due to the similarity in structure shared between IGFs and insulin,binding of insulin to vitronectin was examined. Crosslinking experimentsperformed by Upton et al., 1999, supra indicated that insulin wasunlikely to compete with IGF-II for binding to vitronectin, as was thecase with IGF-I. As a consequence, high concentrations of insulin wereexamined in order to establish whether insulin could compete withradiolabelled IGF-II for binding to vitronectin. The results shown inFIG. 1 indicated that, as for the situation originally observed withIGF-I (Upton et al., 1999, supra), insulin competed poorly with[¹²⁵I]-IGF-II for binding to vitronectin.

Investigation of IGF-I as a competitor for IGF-II binding to vitronectinrevealed that IGF-I, could compete with the binding of [¹²⁵I]-IGF-II tovitronectin (FIG. 2). However, IGF-I was markedly less effective thanIGF-II in competing with radiolabelled IGF-II for binding tovitronectin, requiring an approximate 3000 fold increase in IGF-Iconcentration above IGF-II to achieve the same effect.

Similar studies demonstrated that proIGF-II could compete with[¹²⁵I]-IGF-II for binding to vitronectin (FIG. 3). ProIGF-II was not aseffective as IGF-II in competing with [¹²⁵I]-IGF-II for binding tovitronectin with IC₅₀ values of 65.6 nM and 9.6 nM respectively. Thus,the presence of the E domain within proIGF-II represents a structuralmodification that alters the binding to vitronectin.

This could be due to steric hindrance by the additional domain inproIGF-II compared to IGF-II, or may be a result of an altertedstructure of the proIGF-II molecule that alters the affinity ofproIGF-II for vitronectin.

EXAMPLE 2 Investigation of PAI-1 and uPAR as Competitors for Binding ofIGF-II to Vitronectin

PAI-I and suPAR were investigated as molecules which might compete withIGF-II for binding to vitronectin, as these proteins have been reportedto bind vitronectin (Declerck et al., 1988, J. Biol. Chem. 263 15454;Wei et al.,1994, J.

Biol. Chem. 269 32380; Kanse et al.,1996, Exp. Cell Res. 224 344). Atconcentrations up to 2000 nM competition of PAI-1 with ¹²⁵I-IGF-II forbinding to vitronectin was observed with an approximate IC₅₀ value of524 nM (FIG. 4). While this concentration is relatively high in terms ofIC₅₀ values, such concentrations can be found in vivo associated withtumors (Grondahl-Hansen et al.,1993, Cancer Res. 53 2513). Indeed,Kjoller et al.,1997, Exp. Cell Res. 232 420, found 370 μM of PAI-I wasrequired to achieve half maximal effect in an assay assessing theability of PAI-1 to inhibit cell migration of WISH cells. Thisinhibition was postulated to result via PAI-1 competing with integrinsand uPAR for binding to vitronectin. Thus, the observed interactionbetween PAI-I, IGF-II and vitronectin may indeed have some functionalconsequences in vivo, particularly when IGF-II and PAI-1 levels arehighly expressed as is sometimes found in tumors.

While it is known that PAI-1 binds to the N-terminal somatomedin Bdomain of vitronectin, the high concentrations of PAI-1 required toeffectively compete with IGF-II for binding to vitronectin suggestedthat these studies have not provided any clear information regarding thelocation of IGF-II of the binding site on vitronectin. However, theresults may be interpreted two ways. The first possibility is that theobserved competition between IGF-II and PAI-1 is due to partialcompetition at the primary high affinity site on vitronectin within thesomatomedin B domain via steric hindrance. Alternatively, thecompetition could be due to direct competition between IGF-II and PAI-1,for binding to a site on vitronectin to which PAI-1 binds with a reducedaffinity. Further experimental studies are required to clarify thisissue. While the present inventors were unable to show that solublesuPAR competes for binding of IGF-II to VN, this may have been due tothat fact that lyophilized suPAR was used after internationaltransportation. There is no evidence that reconstituted suPAR isbiologically active after reconstitution, although the observation that[¹²⁵I]-suPAR did not bind VN (data not shown) perhaps supports aninterpretation that the suPAR used in these studies was inactive.

Use of similar techniques to those that identified the PAI-1 bindingsite on vitronectin to be within a fragment of vitronectin containingthe 44 N-terminal amino acids (Deng et al., 1995, Thromb. Haemost. 7466), may establish whether the competition observed between PAI-1 andIGF-II for binding vitronectin is a result of either of thesepossibilities.

It has previously been shown that the soluble form of the urokinasereceptor (suPAR) can bind to immobilized vitronectin (Wei et al., 1994,supra). In the studies reported here, however, even at concentrations upto 300 nM, no competition between suPAR and [¹²⁵I]-IGF-II for binding tovitronectin was observed.

EXAMPLE 3 Binding of IGF-I and IGF-II to Vitronectin in the Presence ofNon-glycosylated Recombinant IGFBP-3

To investigate whether IGFBPs could mediate binding of the IGFs tovitronectin, the effect of increasing concentrations of IGFBP-3 in thepresence of [¹²⁵I]-IGF-I or [¹²⁵I]-IGF-II was assessed. The results areshown in FIG. 5.

At the concentrations tested, 10 ng of IGFBP-3 per well caused thegreatest amount of [¹²⁵I]-IGF-I to bind to vitronectin coated wells,whereas 30 ng of IGFBP-3 per well caused the greatest amount of[¹²⁵I]-IGF-II to bind to vitronectin coated wells. The effect was mostnoticeable in the case of [¹²⁵I]-IGF-I, since low counts of [¹²⁵I]-IGF-Ibound to vitronectin, and even at the lowest concentration of IGFBP-3(3.7 ng), this binding was increased (FIG. 5A). Conversely,[¹²⁵I]-IGF-II did not show an increase over binding obtained onvitronectin coated wells alone, until concentrations of 11 ng/100 μL-33ng/100 μL were reached (FIG. 5B).

Preincubation of IGFBP-3 with either [¹²⁵I]-IGF-I or [¹²⁵I]-IGF-II didnot alter the binding to vitronectin as compared to the nonpre-Incubated concentrations of IGFBP-3 and [¹²⁵I]-IGF-I or[¹²⁵I]-IGF-II.

EXAMPLE 4 Binding of IGF-I to Vitronectin in the Presence of RecombinantIGFBPs Produced in Mammalian Cells

Binding assays examining the ability of [¹²⁵I]-IGF-I to bind toVN-coated dishes in the presence of IGFBPs, added at the same time asthe radiolabel, were performed as described in Upton et al., 1999,supra. The data are shown in FIG. 6. Increasing amounts of IGFBP-2, -4and -5 resulted in increased binding of labelled IGF-I to vitronectincoated wells. At the highest amount of IGFBP tested, 5 ng, binding oflabelled IGF-I was increased approximately 2.8-, 3.8- and 8-fold forIGFBP-2, 4 and 5 compared to control wells where VN, but no IGPBPs, werepresent.

The presence of IGFBP-3 at 0.05, 0.2 and 0.5 ng/well also increasedbinding of labelled IGF-I to VN while 2 and 5 ng of IGFBP-3/wellappeared to inhibit binding of the radiolabel. All concentrations ofIGFBP-1 and -6 tested were also inhibitory.

These results demonstrate that unlike the situation with IGF-II, minimaldirect binding of IGF-I to VN is observed. However, the presence ofIGFBPs, especially IGFBP-2, -3, -4 and -5 enhances IGF binding toVN-coated wells, suggesting that IGFBPs mediate the binding of IGF-I toVN in this situation. In addition, the data suggests that IGFBPs havethe potential to both enhance and inhibit binding of IGF-I to VN,depending on a) which IGFBPs are present and b) the amount of IGFBPpresent.

EXAMPLE 5 Binding of Labelled IGF-I to VN in the Presence IGFBP-3Variants

Binding assays examining the ability of [¹²⁵I]-IGF-I to bind toVN-coated dishes in the presence of an IGFBP-3 variant in which theputative “heparin binding domain” was mutated (IGFBP-3 HBD) and avariant in which glycosylation sites had been mutated (Non-gly IGFBP-3)were performed as described above. The data are shown in FIG. 7.

The IGFBP-3 glycosylation mutant used in these binding studies had threepotential O-glycosylation sites at positions Asn89, Asn109, Asn172mutated to Ala.

The IGFBP-3 HBD mutant did not enhance binding of [¹²⁵I]-IGF-I toVN-coated dishes, suggesting the heparin binding domain of IGFBP-3 isinvolved in binding IGFBP-3 to VN. Other studies have identified thatamino acid residues outside of the putative “heparin binding domain” areresponsible for IGF-I binding to IGFBP-3, hence it is most likely thatthe results represent decreased binding of IGFBP-3 to VN, rather thandecreased binding of labelled IGF-I to IGFBP-3 (Imai et al., 2000, JBiol Chem 275:18188-18194).

Interestingly, the non-glycosylated IGFBP-3 mutant significantlyenhanced binding of [¹²⁵I]-IGF-I to bind to VN-coated dishes. Thestriking 20-fold increase in binding of labelled IGF-I to VN-coatedwells in the presence of 2 ng of the mutant IGFBP-3 is intriguing andsuggests that glycosylation of IGFBP-3 inhibits interaction of either a)IGF-I to IGFBP-3 or b) IGFBP-3 with VN. Alternatively, both interactionsmay be hindered by the presence of carbohydrates.

Whether non-glycoslyated IGFBP-3 is functionally relevant in vivoremains to be established. Nevertheless, this finding suggests thatnon-glycosylated IGFBP-3 bound to VN may be a useful way to deliverIGF-I to sites where IGF-I is required to potentiate cell function suchas in stimulating cell proliferation. Alternatively, non-glycosylatedIGFBP-3 bound to VN may provide a mechanism for sequestering excessIGF-I in situations where cell proliferation is not required such as intumors overexpressing IGFs.

EXAMPLE 6 Competition Binding Assay Assessing Ability of IGF Variants toCompete with Labelled IGFs for Binding to VN in the Presence ofNon-glycosylated Recombinant IGFBP-3

The concentration of IGFBP3 that produced the highest binding ofradiolabelled IGF depicted in FIG. 5 was used in a competition assay todetermine whether increasing concentrations of IGFs and desIGFs couldcompete with radiolabelled IGFs for binding to vitronectin in thepresence of IGFBP3. The results indicated that IGF-I, IGF-II,des(1-6)IGF-II and perhaps also des(1-3)IGF-I (not shown), at highconcentrations, can compete with the binding of [¹²⁵I]-IGF-II tovitronectin in the presence of 10 ng of IGFBP3 (FIG. 8). Theseexperimental results indicated that only IGF-I,-II and des(1-6) IGF-IIcould compete with IGF-II for binding to vitronectin coated wells in thepresence of 30 ng of IGFBP3.

EXAMPLE 7 Stimulation of Cell Proliferation by Isolated ProteinComplexes Comprising IGF-II and Vitronectin

The strategy of pre-binding IGF-II to VN was used in this study in anattempt to more accurately reflect the extracellular environment invivo. Most cell culture approaches add exogenous substrates in solutionphase; thereby the cells are constantly exposed to the treatments. Cellsin tissues do not encounter this “constant, solution phase” environmentin vivo. Thus the approach adopted for this study was to pre-bind IGFsto VN, a situation which more accurately mirrors the in vivo conditions.

Following pre-binding of IGFs to VN in culture dishes, cells were seededinto wells and the, ability of IGFs complexed to VN to stimulate proteinsynthesis was examined by established methods (Francis et al., 1986,Biochem J. 233:207-213). Increased protein synthesis correlates withincreases in cell number, hence is a reflection of cell proliferation.Responses, expressed as percentage above control wells in which no VN orIGFs were present, measured the incorporation of [³H]-leucine into newlysynthesised protein over 48 hrs.

Referring to the data in FIG. 9, when 3, 10, 30, 100, 300 and 1000 ng ofIGF-II was pre-bound to the wells in the absence of VN, resultingresponses of 8, 12, 10, 16, 24 and 43% above the control wells (-VN,-IGF) respectively were observed. Furthermore, these same doses ofIGF-II pre-bound to VN coated wells stimulated incorporation of[³H]-leucine into protein with effects of 19, 29, 39, 51, 70 and 101%respectively. Combining the responses obtained with VN alone (12%) withthat obtained for IGF-II alone (refer to above) gives rise to predictedadditive effects of 20, 24, 22, 28, 36 and 55% for 3, 10, 30, 100, 300and 100 ng of IGF-II respectively. These values are significantlydifferent (p<0.05) to the actual effects observed when IGF-II waspre-bound to VN at all doses except for the two lowest amounts of IGF-Htested (3 and 10 ng). Thus IGF-l pre-bound to VN stimulates synergisticeffects in protein synthesis ranging from 5 to 46% greater than thecalculated additive effects. These responses may well be a result of thedirect binding of IGF-II to VN, and may also arise from indirect bindingof IGF-II to VN via IGFBPs. HaCAT keratinocytes produce large amounts ofIGFBP-3 (Wraight et al., 1994, J. Invest. Dermatol 103:627-631).

EXAMPLE8 Binding of Labelled IGF-H to VN in the Presence of RecombinantIGFBPs Produced in Mammalian Cells

Binding assays examining the ability of [¹²⁵I]-IGF-II to bind toVN-coated dishes in the presence of IGFBPs, added at the same time asthe radiolabel, were performed as described in Upton et al., 1999,supra. The IGFBPs used in these studies were glycosylated having beenproduced in mammalian cells. As shown in FIG. 10, increasing amounts ofIGFBP-1, -3 and -6 resulted in decreased binding of labelled IGF-II tovitronectin coated wells in a dose-dependent manner. IGFBP-2 alsoappeared to compete for binding of labelled IGF-II to VN, albeit lesseffectively than IGFBP-1, -3 or -6. IGFBP-4 on the other hand had littleeffect on binding of IGF-II to VN, while IGFBP-5 appeared to enhanceIGF-II binding to a small extent. The inhibitory effect of IGFBP-3 couldresult from IGFBP-3 competing for binding of IGF-II to the same bindingregion on VN. Alternatively, or in addition, the inhibitory effect ofIGFBP-3, as well as IGFBP-1 and -6. may arise from the affinity ofIGF-II for VN being less than the affinity of IGF-II for these IGFBPs,hence these IGFBPs sequester IGF-II and the complex does not bind to VN.

EXAMPLE 9 Stimulation of Cell Proliferation by Isolated ProteinComplexes Comprising IGF-I, IGFBP-5 and Vitronectin

Referring to Table 2, IGF-I with IGFBP-5 and vitronectin stimulatedkeratinocyte proliferation (as measured by ³H-leucine incorporation intonewly synthesized protein) at all concentrations tested with synergisticeffects observed at the highest amount tested.

It is proposed by the present inventors that the effect of isolatedprotein complexes upon cell proliferation may be even greater at higherconcentrations of IGFBP-5 than the relatively low amount described inTable 2.

EXAMPLE 10 Engineering IGF and IGFBP Variants

Amino acid residues in IGFBP-3 that are important for association withthe ECM and have been defined as the putative “heparin-binding domain”are residues KGRKR at positions 228-232. Similar residues are found inthe corresponding region of IGFBP-5. The IGFBP-3 HBD mutant that wasused in the binding studies reported herein had the residues KGRKRaltered to MDGEA based on the amino acids found in the correspondingpositions in IGFBP-1. These changes result in a charge reversal in thispart of the protein. This mutant still binds IGF-I and IGF-II with highaffinity but binds the acid-labile subunit and the cell surface poorly(Firth et al., 1998, J. Biol. Chem. 273 2631-2638).

Heparin binding motifs in a diverse range of proteins were originallydescribed in Cardin et al., 1989, Arteriosclerosis 9 21-32.

The C-domain of human IGF-II contains a number of positively chargedamino acid residues and in particular positions 34-40 contains the aminoacids RVSRRSR. Given that positively charged amino acids are importantin mediating binding IGFBPs to cell surfaces and to VN, these aminoacids in IGF-II may be important in binding of IGF-II directly to VN.Regardless, it would be a relatively simple procedure to introduce a“heparin binding motif” similar to that found in IGFBP-3 (BXBBB; where Bis a basic amino acid) by creating an IGF-II mutant with deletions ofeither V³⁵ or S³⁶ along with S³⁹. The importance of positive residues inmediating binding of IGF-II to VN is further illustrated by the presentinventors' evidence of reduced binding of the chicken IGF-II mutant,(desR⁴⁰)-IGF-II, to VN.

The C-domain of IGF-I on the other hand contains a relativelynon-charged stretch of amino acids in the corresponding region of theprotein to that described above for IGF-II. This may explain why IGF-Idoes not bind directly to VN. In addition, insulin, which also does notbind to VN (or to IGFBPs) does not have a corresponding C-domain as itis cleaved out in the mature protein. Human IGF-I SSSRRAPQT Human IGF-IIRVSRRS--R

Introduction of the IGF-II sequence RVSRRSR or the IGFBP3 sequence KGRKRinto IGF-I could enable IGF-I to bind VN directly.

EXAMPLE 11 Isolated protein Complexes, Cell Proliferation and Survival

Bc1-2 transcription, a critical element of the cell survival pathway, iselevated in cells that attach to VN through alphav-beta3 integrins.(Matter & Ruoslahti, 2001, 2 3 J. Biol. Chem. 276 27757-27763). Inaddition, IGF-I protects cells from apoptosis by elevating bc1-2transcription in an AKT-dependent manner (Pugazhenthi et al.,1999,. JBiol. Chem. 274 27529-35). The IGF receptor physically associates withthe alphav-beta3 integrin with a synergistic effect on cell growth(Schneller et al.,1997, EMBO J 16 5600-5607). Thus isolated proteincomplexes of the invention may provide an extracellular point ofintegration for initiating the cell survival signals mediated by boththe integrin and the growth factor receptor.

IGFBP-5 has been demonstrated to potentiate the anti-apoptotic andmitogenic effects of IGF-I in prostate cancer cells (Miyake et al.,2000, Endocrinol. 141 2257-2265). In addition, the synthesis of VN invivo by glioma cells and in colorectal adenocarcinoma correlates withtumor grade (Uhm et al., 1999, Clin Cancer Res. 5 1587-1594;Tomasini-Johansson et al., 1994, Exp Cell Res. 214 303-312; Gladson etal., 1995, J. Cell Sci. 108 947-56; Gladson & Cheresh, 1991, J. Clin.Invest. 88 1924-32).

Hence, according to the present invention, it is proposed thatIGF:IGFBP:VN complexes formed in vivo may promote tumor cell survivaland progression. The invention therefore contemplates therapeutic agentsthat disrupt in vivo complex formation.

The heparin-binding domain of VN has been reported to inhibitfibronectin matrix assembly (Hocking et al., 1999, J. Biol. Chem. 27427257-27264). Reduced fibronectin deposition is associated with tumorcell invasion as decreased cell migration rates are associated withincreased levels of polymerised fibronectin (Morla et al., 1994, Nature367 193-196). Hence, IGF:IGFBP complexes bound to the heparin bindingdomain of VN, may dampen fibronectin matrix assembly and facilitatetumor invasion of local connective tissue. Thus, the inventioncontemplates therapeutic agents that disrupt these in vivo complexes toreduce tumor invasiveness.

Isolated Protein Complexes and Wound Healing

The converse argument can be used to support the use of the complex insituations where cell migration is required such as in wound repair. TheIGF system plays an important role in wound healing and both IGF-I andIGFBP-3 are present in wound fluid in significant concentrations.(Skottner et al., 1990, Acta Scand. Suppl. 367 63-66; Clark R (ed) 1996,Molecular and Cell Biology of Wound Repair, pp 3-50, Plenum Press, NewYork; Robertson et al., 1996, Endocrinol. 137 2774-2784; Vogt etal.,1998, Growth Horm. IGF Res. 8 Suppl B:107-9.

IGFBPs have been demonstrated to reduce the rate of IGF clearance fromwounds. (Robertson et al., 1999,. Am J Physiol. 276 E663-71).IGFBP-3:IGF-I complexes bind to fibrin clots in vitro leading to thesuggestion this also occurs in vivo, resulting in concentration of IGF-Iat wound sites. (Campbell et al., 1999, J. Biol. Chem. 274 30215-30221).Similarly, vitronectin binds to fibrin (Podor et al., 2000, J. Biol.Chem. 275 19788-19794). It is also noted that vironectin-null miceexhibit increased wound fibrinolysis and decreased microvascularangiogenesis (Jang et al., 2000, Surgery 127 696-704).

According to the present invention, it is proposed that IGFs bound toIGFBPs can bind to VN, which in turn associates with the fibrin clot,thus providing a reservoir of IGFs at the wound site. Thus isolatedprotein complexes of the invention could be administered to wounds toaccelerate the repair process.

A particular aspect of would healing contemplated by the presentinvention relates to healing diabetic foot ulcers. Wound healing isdelayed in diabetes. Growth factors influence the healing process and inparticular, IGFs have been shown to stimulate keratinocyteproliferation. However, analysis of tissues from diabetic skin and footulcers reveals lack of expression of IGF-I within the basal layer andfibroblasts compared to tissue sections from non-diabetic patients.(Blakytny et al., 2000, J. Pathol. 190 589-594). Isolated proteincomplexes of the invention could be useful in delivery of IGF-I to thesetypes of wounds.

Isolated protein complexes and bone engineering IGFBP-5 facilitatesbinding of labelled IGF-I to bone by a mechanism that is independent ofIGF receptors. (Mohan et al.,1995,. J. Biol. Chem 270 20424-20431) andenhances IGF-stimulated osteoblast function. (Andress, 1995, J. Biol.Chem. 270 28289-28296).

The implant material hydroxylapatite has been shown in numerous studiesto be highly biocomaptible and to osseointegrate well with existingbone.

Recent evidence has found that hydroxylapatite will absorb more VN fromserum than other commonly used implant materials such as titanium andstainless steel. The absorption of VN was accompanied by greater bindingof osteoblast precursor cells (Kilpadi et al., 2001, J. Biomed. Mater.Res. 57 258-267).

The effect of VN on nanophase alumina vs conventional alumina wasrecently examined with VN being found to enhance osteoblast adhesion(Webster et al., 2001, Tissue Eng 7 291-301). The present inventorspropose that isolated protein complexes of the invention may useful ascoating applied to these materials and thereby accelerate bone cellattachment, growth and integration in orthopaedic applications such aship joint replacements.

VN enhances IGF-I stimulated osteoclastic resorption and proteinaseactivities in rabbit bone cell culture. (Rousselle et al., 2001,Histology & Histopathology 16 727-734) and IGFBP-5 enhancesIGF-stimulated osteoblast mitogenesis. (Andress & Birnbaum, 1992, J.Biol. Chem. 267 22467-22472).

The present inventors propose that as the major IGFBP produced by bonecells is IGFBP-5, the potentiation of the IGF effect by VN is likely toalso involve IGFBP-5.

Isolated Protein Complexes and Treatment of Atherosclerosis

IGF-I has previously been implicated in the development of experimentalatherosclerotic lesions. Moreover, alphav-beta3 inhibitors have beendemonstrated to reduce atherosclerotic lesions—this being associatedwith inhibition of IGF-I-mediated signaling. (Nichols et al, 1999, Circ.Res. 851040-1045).

Given that:

(i) IGFBP-5 and VN are synthesised and secreted by arterial smooth.muscle cells and are present in blood vessel walls; and

(ii) IGF:IGFBP-5:VN complexes promote vascular smooth muscle cellmitogenesis and migration (Nam & Clemmons, 2000, Growth Horm. IGF Res.10:A23);

it is proposed by the present inventors that IGF:IGFBP-5:VN complexesare involved in formation of atherosclerotic lesions. Thus therapeuticagents that disrupt complex formation may hold potential in treatingatheroslerosis.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. It will therefore beappreciated by those of skill in the art that, in light of the instantdisclosure, various modifications and changes can be made in theparticular embodiments exemplified without departing from the scope ofthe present invention.

It will also be appreciated that all patent and scientific literatureand computer programs referred to are incorporated herein by reference.TABLE 1 Nucleic acid Reference IGF-I Jansen et al., 1983, Nature 306 609IGF-II Jansen et al., 1985, FEBS Lett 179 243 IGFBP-1 Brinkman et al.,1988, EMBO J. 7 2417 IGFBP-2 Binkert et al., 1989, EMBO J. 8 2497IGFBP-3 Wood et al., 1988, Mol. Endocrinol. 2 1176 IGFBP-4 LaTour etal., 1990, Mol. Endocrinol. 4 1806 IGFBP-5 Kiefer et al., 1991, Biochem.Biophys. Res. Comm. 176 219 IGFBP-6 Shimasaki et al., 1991, Mol.Endocrinol. 5 938 ALS Leong et al., 1992, Mol. Endocrinol. 6 870Vitronectin Suzuki et al., 1985, EMBO J. 4 2519

TABLE 2 Treatment −Vitronectin +Vitronectin Control  100 ± 2.6 — IGFBP-5— 109.5 ± 9.2 100 ng IGF-1 + IGFBP-5 117.4 ± 10.2 119.4 ± 1.9 300 ngIGF-1 + IGFBP-5 140.9 ± 2.0  154.3 ± 1.8 1000 ng IGF-1 + IGFBP-5 144.3 ±11.0 161.4 ± 9.1

1-43. (canceled)
 44. A method of producing an isolated protein complexcomprising IGF-I, an insulin like growth factor binding protein (IGFBP)and vitronectin, said method including the sequential steps of bindingsaid IGFBP to vitronectin and binding IGF-1- to said IGFBP.
 45. A methodof producing an isolated protein complex comprising IGF-I, an insulinlike growth factoring binding protein (IGFBP) and vitronectin, saidmethod including the step of binding said IGFBP with IGF-I boundthereto, to vitronectin.
 46. The method of claim 44, wherein the IGFBPis IGFBP3 or IGFBP5.
 47. The method of claim 46, wherein the IGFBP isIGFBP3.
 48. The method of claim 44, wherein vitronectin is monomeric.49. The method of claim 48, wherein vitronectin is multimeric.
 50. Themethod of claim 44, wherein vitronectin is bound or immobilized to asubstrate.
 51. An isolated protein complex comprising IGF-I, an insulinlike growth factor binding protein (IGFBP) and vitronectin, producedaccording to the method of claim
 44. 52. A surgical implant orprosthesis comprising the isolated protein complex of claim 51.